A method for promoting stable production of bovine embryo development exosomes and application thereof

Abstract

The present application relates to the technical field of exosome, and particularly discloses a stable production method of exosome for promoting bovine embryo development and application thereof. The exosome is prepared by a bovine endometrial epithelial cell line or a bovine oviduct epithelial cell, and stable, large and pure exosome can be obtained, which can be used for large-scale production. Meanwhile, the addition of 100 ug / ml of exosome into the culture medium at the 16-cell stage of bovine embryo development can significantly improve the blastocyst rate and blastocyst quality, and provide support for in vitro culture of embryos.

Description

Technical Field

[0001] This invention belongs to the field of exosome technology, specifically relating to a method for the stable production of exosomes that promote bovine embryonic development and its application. Background Technology

[0002] In the field of mammalian embryo engineering, in vitro embryo culture is an indispensable foundation and core step. Whether produced through in vitro fertilization (IVF), gene editing, or somatic cell nuclear transfer (SCNT) techniques, embryos must undergo an in vitro culture process to develop to a suitable stage (such as the blastocyst stage) before being transferred to a recipient animal for further development. Numerous studies have confirmed that the in vitro culture environment of early embryos has a profound impact on their developmental outcomes, directly determining not only embryonic developmental quality and rate but also significantly affecting implantation efficiency, early pregnancy maintenance, and normal fetal development after embryo transfer. Therefore, optimizing in vitro culture systems and precisely mimicking the in vivo developmental microenvironment are crucial for improving the efficiency and reliability of the entire embryo engineering technology.

[0003] Exosomes, nanoscale extracellular vesicles secreted by cells and carrying specific proteins, nucleic acids, and lipids, have attracted widespread attention in recent years. Studies have shown that exosomes derived from bovine follicular fluid or bovine uterine fluid can be taken up by embryos, effectively promoting their in vitro development and improving embryo quality. This provides a promising new strategy for mimicking the in vivo microenvironment and innovating culture systems.

[0004] However, despite the aforementioned studies confirming the enormous potential of exosomes, their current acquisition methods have significant limitations, severely restricting their transformation into stable, standardized clinical applications or large-scale livestock production. These limitations are mainly reflected in: 1. Limited sources and difficult collection: It relies on animal tissues (such as oviducts and uterus) or physiological fluids (such as follicular fluid and uterine lavage fluid) obtained from slaughterhouses. These sources are not only cumbersome and costly to obtain, but are also greatly affected by factors such as individual animal differences, physiological stages, and health conditions, making it difficult to guarantee a stable supply.

[0005] 2. Complex composition and large batch-to-batch variation: The composition of exosomes extracted directly from organisms is highly dependent on the transient physiological state of the donor animal. This results in significant fluctuations in the composition, concentration and biological effects of exosomes between different batches, which seriously affects the reproducibility of experiments and the stability of culture results.

[0006] 3. Inability to achieve standardized and large-scale production: Due to the above limitations, existing methods cannot produce exosome products with clearly defined components and stable activity in a controllable and uniform manner, making it difficult to meet the consistency requirements of core additives for the industrialization and standardized application of embryo engineering.

[0007] Therefore, it is necessary to develop a method that can overcome the above-mentioned defects and achieve stable, uniform, and large-scale preparation of exosomes. Summary of the Invention

[0008] To address the shortcomings and problems of current methods for obtaining exosomes, such as limited sources, difficult collection, complex composition, and inability to achieve standardized and large-scale production, this invention provides a stable production method for exosomes that promote embryonic development and its application.

[0009] This invention provides a method for the stable production of exosomes, which involves preparing exosomes using bovine endometrial epithelial cell lines.

[0010] The above-mentioned method for stable production of exosomes includes the following steps: S1. After thawing, bovine endometrial epithelial cells were seeded into T25 culture flasks and cultured in a medium containing DMEM, 10% FBS, and 1% antibiotics. The cells were passaged when they reached 80% confluence. S2. Digest the cells with 0.25% trypsin preheated to 37℃ for 5 minutes. Under a microscope, the cells can be seen to separate and become round, indicating that digestion is complete. Add 10% FBS medium to stop digestion, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant to obtain the cells. S3. Add 10% FBS medium and pipette the cells to make a single-cell suspension. Pass the cells at a ratio of 1:3 and expand the culture at 37℃ and 5% CO2 saturated humidity. S4. After passage to the 3rd generation, add 10% FBS and culture for 48 hours until the cell density reaches 80%. Digest the cells and then inoculate them at 1×10⁻⁶ cells / year. 6 Density inoculation at 90cm 2 The culture dish was cultured for another 48 hours. The culture medium was aspirated, washed twice with PBS, and then replaced with 10% exosome-free FBS and cultured for another 48 hours. The culture medium was collected and exosomes were extracted using an ultracentrifuge.

[0011] Application of exosomes prepared by the above method in bovine embryonic development.

[0012] The above application involves adding prepared exosomes to the embryo culture medium during the 16-cell stage of embryonic development.

[0013] In the above application, the amount of exosomes added is 100 ug / ml.

[0014] The application of exosomes prepared by any of the above methods in the preparation of in vitro embryo culture products. This invention provides an in vitro embryo culture method in which 100 μg / ml of exosomes prepared by the above method is added to the basal culture medium when the embryo develops to the 16-cell stage.

[0015] The present invention also provides another method for the stable production of exosomes, which involves the preparation of exosomes using bovine oviduct epithelial cells.

[0016] The above-mentioned method for stable production of exosomes includes the following steps: S1. Take multiple fallopian tubes from different samples, freeze the third-generation fallopian tube epithelial cells, and after thawing, inoculate them into DMEM medium for culture. S2. When the density of fallopian tube epithelial cells reaches 70-80%, the culture medium is replaced with DMEM medium without fetal bovine serum for 48-72 h. The obtained cell supernatant is collected into a 50 mL centrifuge tube and the sample is thawed at 37℃. S3. Transfer the sample to a new centrifuge tube and centrifuge at 2000×g, 4℃, for 30 min. S4. Transfer the supernatant after centrifugation to a new centrifuge tube and centrifuge again at 10,000 × g, 4℃, 45 min to remove larger vesicles. S5. Take the supernatant after centrifugation, filter it through a 0.45 μm filter membrane, and collect the filtrate; S6. Transfer the filtrate to a new centrifuge tube, select an ultracentrifuge rotor, and centrifuge at 100,000 × g for 70 min at 4°C. S7. Remove the supernatant, resuspend in 10 mL of pre-cooled 1×PBS, select an ultracentrifuge rotor, and centrifuge again at 4°C, 100,000 ×g for 70 min. S8. Remove the supernatant, resuspend in 100 μL of pre-cooled 1×PBS, take 20 μL for electron microscopy, and store the remaining exosomes at -80℃.

[0017] The above-described method is used to prepare exosomes for bovine embryonic development or for the preparation of in vitro embryo culture products.

[0018] In the above application, the prepared exosomes are added to the embryo culture medium at the 16-cell stage of embryonic development; the amount of exosomes added is 100ug / ml.

[0019] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention produces exosomes using bovine endometrial epithelial cell lines. The exosome miRNAs prepared at different times and in multiple batches are identical, and the concentration of the produced exosomes is as high as 1.5 × 10⁻⁶. 10 The number of exosomes per ml indicates that the method of the present invention can produce stable, large-volume, and pure exosomes, and can be used for large-scale production of exosomes.

[0020] 2. This invention demonstrates through experiments that adding 100 ug / ml of bovine endometrial epithelial cell line exosomes at the 16-cell stage of embryonic development can significantly increase the blastocyst rate and promote embryonic development. Adding at other stages or adding 200 ug / ml did not have this effect. This indicates that the timing and amount of adding bovine endometrial epithelial cell line exosomes to promote embryonic development are crucial, and can effectively solve the problems of low in vitro blastocyst rate and poor developmental capacity, providing technical support for in vitro embryo culture.

[0021] 3. This invention produces exosomes using bovine oviduct epithelial cell lines, and can obtain stable, large-volume, pure and controllable exosomes at different times and in different batches. At the same time, experiments have verified that adding 100ug / ml of bovine oviduct epithelial cell source at the 16-cell stage has a promoting effect on embryo development and can significantly increase the 8-day blastocyst rate. Attached Figure Description

[0022] Figure 1 The identification results of bovine endometrial epithelial cell line-derived exosomes prepared by the method of the present invention are shown in Figure A, which is the transmission electron microscopy result of exosomes; and Figure B is the nanoparticle size distribution curve of exosomes. Figure 2 The results of Western blot identification of bovine endometrial epithelial cell line-derived exosomes in this invention; Figure 3 This is a diagram showing the overlap of the top 100 highly expressed miRNAs from different generations of exosomes derived from bovine endometrial epithelial cell lines in this invention. Figure 4 The results show the effect of adding bovine endometrial epithelial cell line-derived exosomes on blastocyst rate during the 4-8 cell stage in this invention. Figure 5 The results show the effect of adding bovine endometrial epithelial cell line-derived exosomes to the blastocyst rate at the 16-cell stage in this invention. Figure 6 The present invention incorporates bovine endometrial epithelial cell line-derived exosomes to measure cell apoptosis and reactive oxygen species levels. Figure 7 Apoptosis of blastocyst cells on day 8 after adding bovine endometrial epithelial cell line-derived exosomes in this invention; Figure 8 The results of identification of bovine oviduct epithelial cells extracted in this invention are shown in the figure; (a) is a picture of primary oviduct epithelial cells (5X); (b) is CK18 antibody staining; (c) is DAP1 staining; (d) is a merge fusion image. Figure 9 The image shows the identification results of bovine oviduct epithelial cell-derived exosomes prepared by the method of this invention; Figure A shows the transmission electron microscopy results of the exosomes; Figure B shows the nanoparticle size distribution curve of the exosomes. Figure 10The results of Western blot (WB) identification of bovine oviduct epithelial cell-derived exosomes prepared by the method of this invention; Figure 11 This is a diagram showing the overlap of highly expressed miRNAs in different batches of bovine oviduct epithelial cell-derived exosomes prepared by the method of this invention. Figure 12 The results show the effect of adding bovine oviduct epithelial cell line-derived exosomes on blastocyst rate during the 4-8 cell stage in this invention. Figure 13 The results show the effect of adding bovine oviduct epithelial cell line-derived exosomes to the blastocyst rate at the 16-cell stage in this invention. Figure 14 The first 50 miRNAs of bovine endometrial epithelial cell line-derived exosomes and bovine fallopian tube epithelial cell line-derived exosomes prepared according to this invention are shown. Detailed Implementation

[0023] Current bovine in vitro embryo production technology suffers from bottlenecks such as low embryo production efficiency and poor embryo developmental potential, which significantly limit its large-scale application. To address these issues, this invention provides a method for large-scale, stable production of exosomes and its application in in vitro embryo culture. The invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Example 1: Preparation of exosomes from bovine endometrial epithelial cell lines Bovine endometrial epithelial cells (BEND cells) were purchased from the American Type Culture Collection (ATCC® CRL-2398™). After thawing, they were seeded in T25 culture flasks and cultured in DMEM (Invitrogen) + 10% FBS (Gibco) + 1% antibiotics. Cells were passaged when they reached 80% confluence. Cells were digested with 0.25% trypsin at 37°C for 5 min. Under a microscope, the cells were observed to separate and become rounded, indicating successful digestion. Digestion was stopped by adding 10% FBS, followed by centrifugation at 1000 rpm for 5 min, and the supernatant was discarded. Cells were then resuspended in 10% FBS and passaged at a 1:3 ratio. Cells were cultured at 37°C under 5% CO2 saturated humidity. After passage 3, cells were cultured with 10% FBS for 48 h until the cell density reached 80%. Cells were then digested and cultured at 1×10⁶ cells / mL. 6 Density inoculation at 90cm 2 The culture dish was incubated for another 48 hours. The culture medium was then aspirated, washed twice with PBS, and replaced with 10% exosome-free FBS (HyClone) for another 48 hours. The culture medium was then collected and stored at -80°C.

[0025] The specific method for extracting exosomes using an ultracentrifuge is as follows.

[0026] (1) Samples were melted at 37 °C.

[0027] (2) Transfer the sample to a new centrifuge tube and centrifuge at 2000 × g, 4 ℃, for 30 min.

[0028] (3) Carefully transfer the supernatant to a new centrifuge tube and centrifuge again at 10,000×g, 4 ℃, 45 min to remove larger vesicles.

[0029] (4) Take the supernatant, filter it through a 0.45 μm filter membrane, and collect the filtrate.

[0030] (5) Transfer the filtrate to a new centrifuge tube, select an ultracentrifuge rotor, and centrifuge at 4 ℃, 100,000 × g for 70 min.

[0031] (6) Remove the supernatant, resuspend in 10 mL of pre-cooled 1×PBS, select an ultracentrifuge rotor, and centrifuge again at 4 °C, 100,000×g for 70 min.

[0032] (7) Remove the supernatant, resuspend in 100 μL of pre-cooled 1×PBS, take 20 μL for electron microscopy, and store the remaining exosomes at -80℃.

[0033] (8) Exosome characteristics were identified using transmission electron microscopy, nanoparticle tracking, and Western blotting. Results are as follows: Figure 1 and Figure 2 As shown.

[0034] Depend on Figure 1 Transmission electron microscopy (TEM) results show that the sample is saucer-shaped, consistent with the characteristics of exosomes. Figure 1 Results B show that the average particle size of EXO particles is 129.3 nm, and the concentration is 1.5 × 10⁻⁶. 10 The number of exosomes per ml is consistent with the definition of exosomes, which is between 30 and 200 nm in size, further confirming that the exosomes extracted in this experiment are consistent with the characteristics of exosomes in terms of size.

[0035] Depend on Figure 2 As can be seen, the CD9 protein presents a distinct protein band in EXO, with an estimated size of approximately 25 kDa.

[0036] Example 2: To verify the stability of exosomes prepared by the method of the present invention, exosomes were extracted from the culture medium of BEND cells at passages 3, 4, and 5, respectively. miRNAs were sequenced, and the overlap of the top 100 highly expressed miRNAs in the exosomes of passages 3, 4, and 5 of BEND cells was analyzed. The results are as follows: Figure 3 As shown.

[0037] Depend on Figure 3 It can be seen that there is an overlap between the top 100 highly expressed miRNAs in the exosomes of BEND cells in generations 3, 4, and 5. There are a total of 50 highly expressed miRNAs in the three generations, accounting for 38.2%, which is significantly higher than the proportion of generation-specific miRNAs, indicating that the miRNA composition of exosomes in different generations is highly stable.

[0038] The total amount of exosomes secreted by BEND cells from different passages (P3 / P4 / P5) under the same culture conditions and with similar cell numbers is shown in Table 1 below.

[0039]

[0040] As shown in Table 1, under the same culture environment and similar cell numbers, the number of exosomes (NTA) and total protein content (BCA) of different passages were at the same level, with no significant order-of-magnitude differences. This indicates that the function of exosomes secreted by bovine endometrial epithelial cell lines did not change significantly during passage.

[0041] Example 3: Application of bovine endometrial epithelial cell line-derived exosomes in embryo culture Ovaries were obtained from the slaughterhouse in three batches. Follicles of 2-8 mm were aspirated using an aspiration method. The extracted oocytes were then subjected to in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC).

[0042] Using no exosomes as a blank control (addition amount 0), exosomes were added at concentrations of 100 ug / ml and 200 ug / ml to the embryo culture medium (containing NaCl, KCl, KH2PO4, sodium lactate, MgSO4·7H2O, NaHCO3, CaCl2, NAA, EAA, Glu (glutamine), BSA (serum albumin), and FBS (serum)) at the 4-8 cell and 16 cell development stages, respectively. The number of blastocysts and the blastocyst rate at 8 days were observed and counted. The results are shown in Table 2 below. Figure 4 , 5 As shown.

[0043]

[0044] The results show that, compared with the absence of bovine uterine epithelial cell line-derived exosomes, adding 100 ug / ml and 200 ug / ml exosomes at the 4-8 cell stage did not significantly affect the number and rate of blastocysts. However, adding 100 ug / ml exosomes at the 16-cell stage significantly increased the blastocyst rate, indicating that adding bovine uterine cell line-derived exosomes at the 16-cell development stage has a positive effect on improving the blastocyst rate, highlighting the critical timing of exosome addition. Adding 200 ug / ml exosomes at the 16-cell stage did not promote blastocyst development.

[0045] Example 4: Blastocyst Quality Testing Day 8 blastocysts supplemented with 0 ug / ml and 100 ug / ml exosomes were collected. Reactive oxygen species (ROS) and apoptosis were detected using fluorescence. Results are as follows: Figure 6 , 7 As shown in the figure, NC represents cells that were not treated with exosomes, i.e., the control group; EVs represent the group with 100ug / ml exosomes added.

[0046] Depend on Figure 6 and Figure 7 It can be seen that the cellular reactive oxygen species level was significantly reduced in the 100 ug / ml exosome addition group, which significantly reduced cell apoptosis. Exosomes may reduce cell apoptosis by alleviating oxidative stress, thereby improving blastocyst quality.

[0047] Example 5: Preparation of bovine oviduct epithelial cell-derived exosomes (1) Isolation and identification of fallopian tube epithelial cells Primary fallopian tube epithelial cells were isolated from the fallopian tube, cultured, and passaged. The isolated cells were then identified, and the results are as follows: Figure 8 As shown.

[0048] from Figure 8 As shown in Figure (a), at 5x magnification, the cells exhibit the typical morphology of fallopian tube epithelial cells: tightly packed, uniform in shape, and with clear boundaries, indicating that the cells are in good condition and are qualified primary fallopian tube epithelial cells. Figures (b)-(d) show that the vast majority of the isolated cells are epithelial cells, with uniform cell density, intact cell morphology, good condition, and reliable purity.

[0049] (2) Preparation of exosomes Three different fallopian tube samples were collected, and third-generation fallopian tube epithelial cells were cryopreserved. After thawing, the cells were inoculated into DMEM medium for culture. When the fallopian tube epithelial cell density reached 70-80%, the culture medium was replaced with fetal bovine serum-free DMEM medium for 48-72 h. The obtained cell supernatant was collected into 50 mL centrifuge tubes. The samples were thawed at 37°C. The samples were transferred to a new centrifuge tube and centrifuged at 2000 × g, 4°C, for 30 min. The supernatant was transferred to a new centrifuge tube and centrifuged again at 10,000 × g, 4°C, for 45 min to remove larger vesicles. The supernatant was collected and filtered through a 0.45 μm filter membrane, and the filtrate was collected. The filtrate was transferred to a new centrifuge tube and centrifuged at 100,000 × g, 4°C, for 70 min using an ultracentrifuge rotor. Remove the supernatant, resuspend in 10 mL of pre-chilled 1×PBS, and centrifuge again at 100,000 × g for 70 min at 4 °C using an ultracentrifuge rotor. Remove the supernatant, resuspend in 100 μL of pre-chilled 1×PBS, take 20 μL for electron microscopy, and store the remaining exosomes at -80 °C.

[0050] Exosome characteristics were identified using transmission electron microscopy, nanoparticle tracking, and Western blotting. The results are as follows: Figure 9 and Figure 10 As shown.

[0051] Depend on Figure 9 The results show that EXO exhibits a typical cup-shaped morphology with internal depressions and intact vesicle structures, indicating that the EXO extracted in this experiment conforms to the morphological characteristics of exosomes. The average particle size of the EXO particles is 129.3 nm, and the concentration is 1.5 × 10⁻⁶. 11 per ml.

[0052] Depend on Figure 10 Western blot analysis showed that CD9 and TSG101 proteins exhibited distinct protein bands in the EXO, with expected sizes of approximately 25 kDa and 44 kDa, respectively; while no protein band of Calnexin protein was detected in the EXO, further confirming that the EXO extracted in this experiment conforms to the characteristics of exosomes.

[0053] This embodiment also analyzed the miRNA content of exosomes derived from different bovine oviduct epithelial cells, and the results are as follows: Figure 11 As shown.

[0054] Depend on Figure 11 It can be seen that the miRNA overlap rate of BOEC1, BOEC2 and BOEC3 is 84%, indicating that the miRNA components of exosomes from different batches are highly stable, and stable and controllable exosomes can be produced from different batches at different times, laying the foundation for in vitro embryo production.

[0055] Example 6: Application of bovine oviduct epithelial cell-derived exosomes in embryo culture Ovaries were obtained from the slaughterhouse in three batches. Follicles of 2-8 mm were aspirated using an aspiration method. The extracted oocytes were then subjected to in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC).

[0056] Using no exosomes as a blank control (addition amount 0), exosomes were added to the culture medium at concentrations of 100 ug / ml and 200 ug / ml at the 4-8 cell and 16 cell development stages, respectively. The number of blastocysts and the blastocyst rate at 8 days were observed and counted. The results are as follows: Figure 12 , 13 As shown in Table 3 below.

[0057]

[0058] As shown in Table 3, the blastocyst rate at 8 days was lower in the group with 100 ug / ml or 200 ug / ml bovine oviduct epithelial cell-derived exosomes added during the 4-8 cell stage than in the group without exosomes. In the 16 cell stage, the blastocyst rate at 8 days was higher in the group with 100 ug / ml or 200 ug / ml exosomes added, and the blastocyst-promoting effect of adding 100 ug / ml was better. This indicates that adding 100 ug / ml exosomes during the 16 cell stage can effectively promote blastocyst development.

[0059] Example 7: To further analyze the differences between bovine endometrial epithelial cell line-derived exosomes and bovine fallopian tube epithelial cell line-derived exosomes prepared in this invention, this example analyzed the distribution of the top 50 highly co-expressed miRNAs in BEND and BOEC. The results are as follows: Figure 14 As shown.

[0060] Depend on Figure 14 It can be seen that 29 of the top 50 miRNAs in both groups overlap, accounting for 40.8% of the total count. This means that more than half (29 / 50 = 58%) of the top 50 miRNAs in BEND and BOEC are shared, indicating that these miRNAs are highly expressed core members in both cell types and may play fundamental and crucial biological functions. At the same time, BEND and BOEC each have 21 unique top 50 miRNAs, indicating significant specificity in the miRNA expression profiles of the two cell types. These unique miRNAs may be related to the unique functions and phenotypes of their respective cells.

[0061] The above are merely preferred embodiments of the present invention and do not limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for stable production of exosomes, characterized in that: Exosomes were prepared using bovine endometrial epithelial cell lines.

2. The method for stable production of exosomes as described in claim 1, characterized in that: Includes the following steps: S1. After thawing, bovine endometrial epithelial cells were seeded into T25 culture flasks and cultured in a medium containing DMEM, 10% FBS, and 1% antibiotics. The cells were passaged when they reached 80% confluence. S2. Digest the cells with 0.25% trypsin preheated to 37℃ for 5 minutes. Under a microscope, the cells can be seen to separate and become round, indicating that digestion is complete. Add 10% FBS medium to stop digestion, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant to obtain the cells. S3. Add 10% FBS medium and pipette the cells to make a single-cell suspension. Pass the cells at a ratio of 1:3 and expand the culture at 37℃ and 5% CO2 saturated humidity. S4. After passage to the 3rd generation, add 10% FBS and culture for 48 hours until the cell density reaches 80%. Digest the cells and then inoculate them at 1×10⁻⁶ cells / year. 6 Density inoculation at 90cm 2 The culture dish was cultured for another 48 hours. The culture medium was aspirated, washed twice with PBS, and then replaced with 10% exosome-free FBS and cultured for another 48 hours. The culture medium was collected and exosomes were extracted using an ultracentrifuge.

3. The application of exosomes prepared by the method described in claim 1 or 2 in bovine embryonic development.

4. The application as described in claim 3, characterized in that: The prepared exosomes were added to the embryo culture medium during the 16-cell stage of embryonic development.

5. The application as described in claim 4, characterized in that: The amount of exosomes added was 100 ug / ml.

6. The use of exosomes prepared by the method described in claim 1 or 2 in the preparation of in vitro embryo culture products.

7. A method for in vitro embryo culture, characterized in that: When the embryo develops to the 16-cell stage, 100 μg / ml of exosomes prepared by the method described in claim 1 or 2 are added to the basal culture medium.

8. A method for stable production of exosomes, characterized in that: Exosomes were prepared using bovine oviduct epithelial cells.

9. The use of exosomes prepared by the method of claim 8 in bovine embryonic development or in the preparation of in vitro culture products of embryos.

10. The application as described in claim 9, characterized in that: The prepared exosomes were added to the embryo culture medium at the 16-cell stage of embryonic development; the amount of exosomes added was 100 ug / ml.

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